A Triple Culture Cell System Modeling the Human Blood-Brain Barrier

Abstract

The delivery of drugs to the brain remains a challenge due to the blood-brain barrier's (BBB) highly specific and restrictive properties, which controls and restrict access to the brain parenchyma. However, with the development of nanotechnologies, large panels of new nanomaterials were developed to improve drug delivery, highlighting the need for reliable in vitro microsystems to predict brain penetration in the frame of preclinical assays. Here is a straightforward method to set up a microphysiological system to model the BBB using solely human cells. In its configuration, the model consists of a triple culture including brain-like endothelial cells (BLECs), pericytes, and astrocytes, the three main BBB cellular actors necessary to induce and regulate the BBB properties in a more physiological manner without the requirement of tightening compounds. The model developed in a 12-well plate format, ready after 6 days of triple culture, is characterized in physical properties, gene, and protein expressions and used for polymeric nanogel transport measurement. The model can be used for an extensive range of experiments in healthy and pathological conditions and represents a valuable tool for preclinical assessments of molecule and particle transport, as well as inter-and intracellular trafficking.

Pioglitazone Attenuates the Effects of Peripheral Inflammation in a Human In Vitro Blood–Brain Barrier Model

“…The medium was renewed every 2 days. After this period, ECs acquire a BBB phenotype as previously demonstrated [ 56 , 60 , 61 ] and become BLECs.…”

Astrocytes and human artificial blood-brain barrier models

“…However, the disadvantage of this setting is that the two cell types cannot be readily separated in experimental protocols using Western blotting or transport and accumulation studies [ 77 ]. Given the importance of NVU cells in the development and maintenance of the BBB and the coordinated response to the pathological environment, in vitro models of the BBB should consider incorporating all critical cellular components (BMECs, astrocytes, and pericytes) to enhance the physiological relevance [ 77 , 127 , 130 , 131 ]. The beneficial effect of triple co-cultures and mixed co-cultures has been shown in creating a more efficient in vitro BBB model in terms of high TEER [ 108 , 127 ].…”

“…Furthermore, differentiation of human pluripotent stem cells into BMCEs may lack the functional properties of native hBMECs, which may hinder the development of a robust and physiologically relevant human in vitro BBB model for functional studies and drug discovery [ 138 ]. Recently, Rizzi et al [ 131 ] developed an in vitro microsystem with triple culture configuration to predict brain penetration in preclinical assays. They presented a microphysiological system for modeling the BBB using only human cells, including brain-like endothelial cells, pericytes, and astrocytes.…”

“…A limitation of the model was the lack of shear stress, which has been shown to affect endothelial cell differentiation and TJ protein expression. Nevertheless, the authors demonstrated that they could reproduce the architecture of the human BBB in vitro [ 131 ].…”

“…In addition, it showed sufficient barrier function to study the passage of large molecules and is sensitive to differences in antibody penetration, which could support the discovery and development of BBB shuttle technologies [ 141 ]. In another study, Rizzi et al [ 131 ] developed triple culture in vitro microsystem to predict brain penetration in the frame of preclinical assays. They presented a microphysiological system to model the BBB using solely human cells, including brain-like endothelial cells, pericytes, and astrocytes.…”